Each of the four intracellular cysteines of CD36 is essential for insulin- or AMP-activated protein kinase-induced CD36 translocation.

Stimulation of cellular fatty acid uptake by induction of insulin signalling or AMP-kinase (AMPK) activation is due to translocation of the fatty acid-transporter CD36 from intracellular stores to the plasma membrane (PM). For investigating the role of the four Cys-residues within CD36's cytoplasmic tails in CD36 translocation, we constructed CHO-cells expressing CD36 mutants in which all four, two, or one of the intracellular Cys were replaced by Ser. Intracellular and PM localization of all mutants was similar to wild-type CD36 (CD36wt). Hence, the four Cys do not regulate sub-cellular CD36 localization. However, in contrast to CD36wt, insulin or AMPK activation failed to induce translocation of any of the mutants, indicating that all four intracellular Cys residues are essential for CD36 translocation. The mechanism of defective translocation of mutant CD36 is unknown, but appears not due to loss of S-palmitoylation of the cytoplasmic tails or to aberrant oligomerization of the mutants.

Insulin and chromium picolinate induce translocation of CD36 to the plasma membrane through different signaling pathways in 3T3-L1 adipocytes, and with a differential functionality of the CD36.

Chromium picolinate (CrPic) has been indicated to activate glucose transporter 4 (GLUT4) trafficking to the plasma membrane (PM) to enhance glucose uptake in 3T3-L1 adipocytes. In skeletal and heart muscle cells, insulin directs the intracellular trafficking of the fatty acid translocase/CD36 to induce the uptake of cellular long-chain fatty acid (LCFA). The current study describes the effects of CrPic and insulin on the translocation of CD36 from intracellular storage pools to the PM in 3T3-L1 adipocytes in comparison with that of GLUT4. Immunofluorescence microscopy and immunoblotting revealed that both CD36 and GLUT4 were expressed and primarily located intracellularly in 3T3-L1 adipocytes. Upon insulin or CrPic stimulation, PM expression of CD36 increased in a similar manner as that for GLUT4; the CrPic-stimulated PM expression was less strong than that of insulin. The increase in PM localization for these two proteins by insulin paralleled LCFA ([1-(14)C]palmitate) or [(3)H]deoxyglucose uptake in 3T3-L1 adipocytes. The induction of the PM expression of GLUT4, but not CD36, or substrate uptake by insulin and CrPic appears to be additive in adipocytes. Furthermore, wortmannin completely inhibited the insulin-stimulated translocation of GLUT4 or CD36 and prevented the increased uptake of glucose or LCFA in these cells. Taken together, for the first time, these findings suggest that both insulin and CrPic induce CD36 translocation to the PM in 3T3-L1 adipocytes and that their translocation-inducing effects are not additive. The signaling pathway inducing the translocations is different, apparently resulting in a differential activity of CD36.

Effects of AMPK activators on the sub-cellular distribution of fatty acid transporters CD36 and FABPpm.

In heart and skeletal muscle, enhanced contractile activity induces an increase in the uptake of glucose and long-chain fatty acids (LCFA) via an AMP-activated protein kinase (AMPK)-regulated mechanism. AMPK activation induces glucose uptake through translocation of glucose transporter 4 (GLUT4) from intracellular pools to the plasma membrane (PM). AMPK-mediated LCFA uptake has been suggested to be regulated by a similar translocation of the LCFA transporters CD36 and plasma membrane-associated fatty acid binding protein (FABPpm). In contrast to the well-characterized GLUT4 translocation, documentation of the proposed translocation of both LCFA transporters is rudimentary. Therefore, we adopted a cell culture system to investigate the localization of CD36 and FABPpm compared with GLUT4, in the absence and presence of AMPK activators oligomycin and AICAR. To this end, intact Chinese hamster ovary (CHO) cells stably expressing CD36 or myc-tagged GLUT4 (GLUT4myc) were used; FABPpm is endogenously expressed in CHO cells. Immuno-fluorescence microscopy revealed that CD36 PM localization resembled that of GLUT4, while FABPpm localized to other PM domains. Upon stimulation with oligomycin or AICAR, CD36 translocated (1.5-fold increase) to a PM location similar to that of GLUT4myc. In contrast, the PM FABPpm content did not change upon AMPK activation. Thus, for the first time in intact cells, we present evidence for AMPK-mediated translocation of CD36 from intracellular pools to the PM, similar to GLUT4, whereas FABPpm is not relocated.

Insulin-induced translocation of CD36 to the plasma membrane is reversible and shows similarity to that of GLUT4.

In cardiac and skeletal muscles, insulin regulates the uptake of long-chain fatty acid (LCFA) via the putative LCFA transporter CD36. Biochemical studies propose an insulin-induced translocation of CD36 from intracellular pools to the plasma membrane (PM), similar to glucose transporter 4 (GLUT4) translocation. To characterize insulin-induced CD36 translocation in intact cells, Chinese hamster ovary (CHO) cells stably expressing CD36 or myc-tagged GLUT4 (GLUT4myc) were created. Immuno-fluorescence microscopy revealed CD36 to be located both intracellularly (in--at least partially--different compartments than GLUT4myc) and at the PM. Upon stimulation with insulin, CD36 translocated to a PM localization similar to that of GLUT4myc; the increase in PM CD36 content, as quantified by surface-protein biotinylation, amounted to 1.7-fold. The insulin-induced CD36 translocation was shown to be phosphatidylinositol-3 kinase-dependent, and reversible (as evidenced by insulin wash-out) in a similar time frame as that for GLUT4. The expression of GLUT4myc in non-stimulated cells, and the insulin-induced increase in PM GLUT4myc correlated with increased deoxyglucose uptake. By contrast, CD36 expression in non-stimulated cells and the insulin-induced increase in PM CD36 were not paralleled by a rise in LCFA uptake, suggesting that in these cells, such increase requires additional proteins, or a protein activation step. Taken together, this study is the first to present morphological evidence for CD36 translocation, and shows this process to resemble GLUT4 translocation.

Fic1 is expressed at apical membranes of different epithelial cells in the digestive tract and is induced in the small intestine during postnatal development of mice.

Mutations in ATP8B1 are associated with FIC1 disease, an autosomal recessive disorder in which intrahepatic cholestasis is the predominant manifestation. ATP8B1 encodes FIC1, which is expressed in several tissues, most prominently in the intestine, pancreas, and stomach and, to a much lesser extent, in the liver. In this study, Fic1 localization and expression during postnatal development was examined in healthy mice. Immunoblot and RT-PCR analysis indicated Fic1 is expressed abundantly in regions of the adult gastrointestinal tract of humans and mice. Immunohistochemistry revealed that Fic1 was localized to the apical membranes of enterocytes, pancreatic acinar cells, gastric pit epithelial cells, and hepatocytes and cholangiocytes. Subsequent analysis of early postnatal expression revealed that Fic1 expression in the small intestine was limited or absent at the age of 7 and 14 d and increased significantly with maturation. In contrast, pancreatic, hepatic, and gastric Fic1 expression was not diminished during the first 3 wk of postnatal development. In conclusion, these data show that Fic1 is expressed in a tissue-specific and developmentally regulated fashion at the apical membranes of epithelial cells. We speculate that the developing bile salt pool in the maturing intestine accounts for the increase in Fic1 protein expression in this tissue.

Rabaptin-5alpha/rabaptin-4 serves as a linker between rab4 and gamma(1)-adaptin in membrane recycling from endosomes.

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-adaptin regulate membrane recycling.